White rust of <Emphasis Type="Italic">Ipomoea</Emphasis> caused by <Emphasis Type="Italic">Albugo ipomoeae-panduratae</Emphasis> and <Emphasis Type="Italic">A. ipomoeae-hardwickii</Emphasis> and their host specificity

In some areas of Japan, yellow spots with white pustules on leaves, stems, petioles, peduncles and calyces were found on Ipomoea nil, I. triloba, I. lacunosa and I. hederacea var. integriuscula. We demonstrated that the diseases on I. nil, I. triloba and I. lacunosa were caused by host-specific strains of Albugo ipomoeae-panduratae and defined three forma speciales of the fungus, respectively, for the three Ipomoea species: “f. sp. nile”, “f. sp. trilobae” and “f. sp. lacunosae”. Because the diseases were new to Japan, we coined the Japanese name “shirosabi-byo”, which means white rust. We also showed that the disease on I. hederacea var. integriuscula was caused by A. ipomoeae-hardwickii. We named this new disease “white rust (shirosabi-byo in Japanese)”.     

Toxigenicity and pathogenicity of <Emphasis Type="Italic">Fusarium poae</Emphasis> and <Emphasis Type="Italic">Fusarium avenaceum</Emphasis> on wheat

In a field experiment between 2004 and 2006, 14 winter wheat varieties were inoculated with either a mixture of three isolates of F. poae or a mixture of three isolates of F. avenaceum. In a subsequent climate chamber experiment, the wheat variety Apogee was inoculated with individual single conidium isolates derived from the original poly conidium isolates used in the field. Disease symptoms on wheat heads were visually assessed, and the yield as well as the fungal incidence on harvested grains (field only) was determined. Furthermore, grains were analysed using LC-MS/MS to determine the content of Fusarium mycotoxins. In samples from field and climate chamber experiments, 60 to 4,860 μg kg⁻¹ nivalenol and 2,400 to 17,000 μg kg⁻¹ moniliformin were detected in grains infected with F. poae and F. avenaceum, respectively. Overall, isolate mixtures and individual isolates of F. avenaceum proved to be more pathogenic than those of F. poae, leading to a higher disease level, yield reductions up to 25%, and greater toxin contamination. For F. poae, all variables except for yield were strongly influenced by variety (field) and by isolate (climate chamber). For F. avenaceum, variety had a strong effect on all variables, but isolate effects on visual disease were not reflected in toxin production. Correlations between visual symptoms, fungal incidence, and toxin accumulation in grains are discussed.     

Anthracnose of Christmas rose caused by <Emphasis Type="Italic">Colletotrichum</Emphasis> sp.

Leaf spots were found on Christmas rose (Helleborus niger) in Yamagata Prefecture, Japan, in October 2006. The morphology of the causal fungus was very close to that of Colletotrichum truncatum. Classifying the species from the sequences of the internal transcribed spacer regions of ribosomal DNA was inconclusive, and the isolates were identified only as Colletotrichum sp. Artificial inoculation confirmed the pathogenicity of isolates to the host plant and some legumes. We propose the name anthracnose of Christmas rose for this disease by Colletotrichum sp.     

Vascular blackening of wasabi rhizomes caused by <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis>

Wasabi (Wasabia japonica) is grown for its highly-valued rhizome which is used as a condiment in Japanese food. Symptoms of vascular blackening in the rhizome were first observed in 2005 in plants grown in British Columbia, Canada. Microscopic observations and microbial isolation from infected tissues revealed that most of the xylem tracheid cells were blackened and bacteria were consistently associated with symptomatic plants. The bacterium most frequently recovered was identified as Pectobacterium carotovorum subsp. carotovorum (Pcc) using BioLog™ and sequencing of a specific ~510 bp IGS region. Pathogen-free plants obtained using meristem-tip micropropagation were inoculated with a wasabi isolate of Pcc. Vascular blackening symptoms developed in the rhizome after 8 weeks when the rhizome was first wounded by stabbing or cutting, or if the roots were pre-inoculated with Pythium species isolated from rhizome epidermal tissues, followed by inoculation with Pcc at 1 × 10⁸ cells ml⁻¹. Xylem tracheid cells were blackened and Pcc was reisolated from all diseased tissues. The highest frequency of rhizome vascular blackening occurred at 22°C and 27°C and these tissues occasionally succumbed to soft rot at higher temperatures, but not when inoculated tissues were incubated at 10°C. The rooting medium used by growers for vegetative propagation of wasabi was shown to contain Pcc but the pathogen was not recovered from the irrigation water. Entry of Pcc through wounds on wasabi rhizomes and the host tissue response result in symptoms of vascular blackening.     

Assessment of recent outbreaks of <Emphasis Type="Italic">Dickeya</Emphasis> sp. (syn. <Emphasis Type="Italic">Erwinia chrysanthemi</Emphasis>) slow wilt in potato crops in Israel

Suspected Dickeya sp. strains were obtained from potato plants and tubers collected from commercial plots. The disease was observed on crops of various cultivars grown from seed tubers imported from the Netherlands during the spring seasons of 2004–2006, with disease incidence of 2–30% (10% in average). In addition to typical wilting symptoms on the foliage, in cases of severe infection, progeny tubers were rotten in the soil. Six strains were characterised by biochemical, serological and PCR-amplification. All tests verified the strains as Dickeya sp. The rep-PCR and the biochemical assays showed that the strains isolated from blackleg diseased plants in Israel were very similar, if not identical to strains isolated from Dutch seed potatoes, suggesting that the infection in Israel originated from the Dutch seed. The strains were distantly related to D. dianthicola strains, typically found in potatoes in Western Europe, and were similar to biovar 3 D. dadanti or D. zeae. This is the first time that the presence of biovar 3 strains in potato in the Netherlands is described. One of the strains was used for pathogenicity assays on potato cvs Nicola and Mondial. Symptoms appeared 2 to 3 days after stem inoculation, and 7 to 10 days after soil inoculation. The control plants treated with water, or plants inoculated with Pectobacterium carotovorum, did not develop any symptoms with either method of inoculation. The identity of Dickeya sp. and P. carotovorum re-isolated from inoculated plants was confirmed by PCR and ELISA.     

Fusarium rot of hyacinth caused by <Emphasis Type="Italic">Gibberella zeae</Emphasis> (anamorph: <Emphasis Type="Italic">Fusarium</Emphasis><Emphasis Type="Italic"> graminearum</Emphasis>)

Severe rot of leaves, peduncles and flowers caused by Gibberella zeae (anamorph: Fusarium graminearum) was found on potted plants of hyacinth (Hyacinthus orientalis), a liliaceous ornamental, in greenhouses in Kagawa Prefecture, Japan, in January 2001. This disease was named “Fusarium rot of hyacinth” as a new disease because only the anamorph, F. graminearum, was identified on the diseased host plant. The authors contributed equally to this work. The fungal isolate and its nucleotide sequence data obtained in this study were deposited in the Genebank, National Institute of Agrobiological Sciences and the DDBJ/EMBL/GenBank databases under the accession numbers MAFF239499 and AB366161, respectively.     

Natural mechanisms for cereal resistance to the accumulation of <Emphasis Type="Italic">Fusarium</Emphasis> trichothecenes

This review describes the naturally occurring mechanisms in cereals that lead to a reduction of Fusarium trichothecene mycotoxin accumulation in grains. A reduction in mycotoxin contamination in grains could also limit fungal infection, as trichothecenes have been reported to act as virulence factors. The mechanisms explaining the low toxin accumulation trait, generally referred to as type V resistance to Fusarium, can be subdivided into two classes. Class 1 includes mechanisms by which the plants chemically transform the trichothecenes, leading to their degradation or detoxification. Among the detoxification strategies, glycosylation of trichothecenes is a natural process already reported in wheat. According to the structure and the toxicity of trichothecenes, two other detoxification processes, acetylation and de-epoxidation, can be expressed, at least in transgenic plants. Class 2 comprises mechanisms that lead to reduced mycotoxin accumulation by inhibition of their biosynthesis through the action of plant endogenous compounds. These include both grain constitutive compounds and compounds induced in response to pathogen infection. There are already many compounds with antioxidant properties, like phenolic compounds, peptides or carotenoids, and with prooxidant properties, like hydrogen peroxide or linoleic acid-derived hydroperoxides, that have been described as ‘modulators’ of mycotoxin biosynthesis. This review addresses for the first time different studies reporting specific in vitro effects of such compounds on the biosynthesis of Fusarium mycotoxins. A better understanding of the natural processes limiting accumulation of trichothecenes in the plant will open the way to the development of novel breeding varieties with reduced ‘mycotoxin risk’.     

Characterization of <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis> causing soft-rot disease on <Emphasis Type="Italic">Pinellia ternata</Emphasis> in China

Pinellia ternata is a traditional Chinese herb which has been used in China for over 1,000 years. A soft-rot disease characterized by water-soaked lesions and soft-rot symptoms with a stinking odour was commonly observed in cultivated fields of this plant, and Pectobacterium-like bacteria were consistently isolated from the infected tissues. Two typical strains (SXR1 and ZJR1), isolated from Shanxi and Zhejiang, respectively, were identified. Pathogenicity tests revealed that these strains were virulent to P. ternata and induced the same symptoms as observed in the field. Characterization involving fatty acid profile, metabolic and physiological properties, 16S rDNA sequence and PCR-RFLP identified both isolates as P. carotovorum subsp. carotovorum (Pcc). The 16S rDNA of both isolates shared 97–99% sequence similarity with that of Pcc strains. The phylogenetic trees showed that both isolates were clustered in the group of Pcc and P. carotovorum subsp. odorifera and both PCR-RFLP profiles were consistent with the pattern E produced by the minority of Pcc strains. Thus, isolates SXR1 and ZJR1 were characterized as Pcc in spite of some differences. This is the first report that Pcc has been proven as a causal agent of soft-rot disease on P. ternata.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation for random insertional mutagenesis in <Emphasis Type="Italic">Colletotrichum lagenarium</Emphasis>

Random insertional mutagenesis using a marker DNA fragment is an effective method for identifying fungal genes relevant to morphogenesis, metabolism, and so on. Agrobacterium tumefaciens-mediated transformation (AtMT) has long been used as a tool for the genetic modification of a wide range of plant species. Recent study has indicated that A. tumefaciens could transfer T-DNA not only to plant cells but also to fungal cells. In this study, AtMT was applied to Colletotrichum lagenarium for random insertional mutagenesis. We constructed a binary vector pBIG2RHPH2 carrying a hygromycin-resistant gene cassette between the right and left borders of T-DNA. Optimal co-cultivation of C. lagenarium wild-type 104-T with pBIG2RHPH2-introduced A. tumefaciens C58C1 led to the production of 150–300 hygromycin-resistant transformants per 10⁶ conidia. Southern blot analysis revealed that T-DNA was mainly integrated at a single site in the genome and at different sites in transformants. The T-DNA inserts showed small truncations of either end, but the hygromycin-resistant gene cassette inside the T-DNA was generally intact. The mode of T-DNA insertion described above resulted in highly efficient gene recovery from the transformants by thermal asymmetrical interlaced-polymerase chain reaction. The fungal genomic DNA segments flanking T-DNA were identified from five of eight mutants that had defective melanin biosynthesis. The sequence from one of the segments was identical to that of the melanin biosynthesis gene PKS1 of C. lagenarium, which we previously characterized. These results strongly support our notion that AtMT is a possible tool for tagging genes relevant to pathogenicity in the plant pathogenic fungus C. lagenarium.     

Cytological events in tomato leaves inoculated with conidia of <Emphasis Type="Italic">Blumeria graminis</Emphasis> f. sp. <Emphasis Type="Italic">hordei</Emphasis> and <Emphasis Type="Italic">Oidium neolycopersici</Emphasis> KTP-01

Leaves of tomato and barley were inoculated with conidia of Blumeria graminis f. sp. hordei race 1 (R1) or Oidium neolycopersici (KTP-01) to observe cytological responses in search of resistance to powdery mildew. Both conidia formed appressoria at similar rates on tomato or barley leaves, indicating that no resistance was expressed during the prepenetration stage of these fungi. On R1-inoculated tomato leaves, appressoria penetrated the papillae, but subsequent haustorium formation was inhibited by hypersensitive necrosis in the invaded epidermal cells. On the other hand, KTP-01 (pathogenic to tomato leaves) successfully developed functional haustoria in epidermal cells to elongate secondary hyphae, although the hyphal elongation from some conidia was later suppressed by delayed hypersensitive necrosis in some haustorium-harboring epidermal cells. Thus, the present study indicated that the resistance of tomato to powdery mildew fungi was associated with a hypersensitive response in invaded epidermal cells but not the prevention of fungal penetration through host papilla.     

Evaluation of nematicidal properties of saponins from <Emphasis Type="Italic">Medicago</Emphasis> spp.

The nematicidal activity of saponins from Medicago arborea (tops), M. arabica (tops and roots) and M. sativa (tops and roots) against the plant-parasitic nematode Xiphinema index was investigated. Nematicidal activity of related prosapogenins and sapogenins on X. index is also described. Saponins from Medicago spp. at different concentrations were all nematicidal, those from M. arborea tops being the less effective. In general, saponins induced 100% mortality at 500 μg ml⁻¹ between 8 and 48 h, while prosapogenins resulted in toxicity starting at 125 μg ml⁻¹. Differences in the effects on X. index induced by prosapogenins and sapogenins were less pronounced, although prosapogenins displayed a larger range of activity. Assays with purified sapogenins demonstrated the relationship of the observed nematicidal activity of M. sativa and M. arborea to the content of the main aglycones (medicagenic acid and hederagenin, respectively) in the saponin extracts. Hederagenin displayed the highest bioactivity, giving 38% mortality after 1 h at 125 μg ml⁻¹.     

Detection of viridiofungin A and other antifungal metabolites excreted by <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> active against different plant pathogens

Antibiosis is assumed to be an essential mechanism exerted by potential biocontrol agents (BCAs) of Trichoderma spp. Therefore, in the present study, we report for the first time on the elucidation and production of viridiofungin A (VFA) from T. harzianum isolate T23 cultures and investigate the antifungal potential of VFA and some other secondary metabolites purified from T. harzianum cultures against Fusarium moniliforme. The bioautography assay revealed that T. harzianum isolates T16 and T23 excreted several secondary metabolites with antifungal activity. Following isolation and purification of the antifungal zones, three fractions (F223, F323 and F423) from extracts of isolate T23 and two fractions (F416 and F516) from extracts of isolate T16 exhibited pronounced fungitoxic activity in the bioautography and antibiotic disk assays against Cladosporium spp. and F. moniliforme, respectively. The structure of the antifungal metabolite in fraction F323 was identified as viridiofungin A (VFA), the first report of production of VFA by isolate T23 of T. harzianum. Following cultivation of isolate T23 in PDB medium for 9 days, 94.6 mg l⁻¹ of VFA were determined. VFA and fraction F516 retarded the mycelial growth of F. moniliforme in the non-volatile phase assay by >90% for each 250 μg ml⁻¹ 7 days post-inoculation (dpi). While VFA and fraction F416 showed both volatile and non-volatile effects, fraction F516 seemed to exhibit mainly non-volatile activity. Microscopic examination revealed that hyphae of F. moniliforme grown on VFA-amended medium were less branched and appeared thicker than untreated hyphae. Furthermore, in the presence of VFA, formation of chlamydospores by F. moniliforme was increased. Finally, the antifungal spectrum of VFA towards various important plant pathogens was evaluated. Germination of propagules of a variety of fungal pathogens in vitro was differentially inhibited by VFA. While in the presence of 100 μg ml⁻¹ VFA conidial germination of V. dahliae was completely inhibited, a slightly higher concentration (150 μg ml⁻¹) of the inhibitor was required to suppress germination of Phytophthora infestans sporangia or sclerotia of Sclerotinia sclerotiorum. Contrary to several reports in the literature, VFA proved to be fungistatic rather than fungicidal. However, neither VFA nor the other Trichoderma metabolites, such as 6PAP, F416 and F516, exhibited any antibacterial activity against Gram-positive and Gram-negative bacteria.     

A promising strain of endophytic <Emphasis Type="Italic">Streptomyces</Emphasis> sp. for biological control of cucumber anthracnose

In a survey for effective biocontrol agents of Colletotrichum orbiculare, causal agent of cucumber anthracnose, 43 (MBCu series) and 135 (MBPu series) endophytic actinomycete strains were recovered from surface-sterilized organs of cucumber (Cucumis sativus) and pumpkin (Cucurbita moschata) plants, respectively. The strains were cultured with C. orbiculare on IMA-2 agar medium to determine their in vitro antagonistic ability. Eleven strains that strongly inhibited hyphal growth of the pathogen were selected as potent antagonists. Detached cotyledons of cucumber were soaked in a spore suspension of an antagonistic strain 1 day before challenge-inoculation with the pathogen, and six of these antagonists (MBCu-32, 36, 42, 45, and 56, and MBPu-75) significantly reduced the number and size of the lesions on the cotyledons compared to the untreated control. In the same way, these six strains inhibited lesion development on attached leaves of 3-week-old cucumber seedlings. Strain MBCu-56, the most suppressive of the strains, was selected for further tests. Its suppressiveness increased as concentrations increased; pretreatment of leaves with the strain at 10⁷, 10⁸ and 10⁹ cfu/ml suppressed disease by 72, 79 and 93%, respectively. These results strongly indicated that MBCu-56 has strong potential for controlling cucumber anthracnose. Based on the taxonomic characteristics and 16S rDNA sequence, MBCu-56 was identified as Streptomyces sp. With scanning electron microscopy, the substrate mycelia of the strain were seen to colonize the surface of leaves above the cuticle. Some hyphae also penetrated and grew underneath the cuticle.     

<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimethylsphingosine,an inhibitor of sphingosine kinase,induces phytoalexin production and hypersensitive cell death of Solanaceae plants without generation of reactive oxygen species

Plant recognition of elicitors derived from pathogens induces various resistant reactions, including production of reactive oxygen species, hypersensitive cell death and accumulation of phytoalexins. Previously, we isolated a ceramide elicitor from Phytophthora infestans, which activates O₂ ⁻ production of potato suspension-cultured cells. In this study, we employed nine ceramide-related chemicals to test their elicitor activity. Although, none of the tested chemicals induced O₂ ⁻ production, N,N-dimethylsphingosine (DMS) induced accumulation of phytoalexin in potato tubers. In potato, tobacco and Nicotiana benthamiana, DMS also induced rapid cell death. DMS-treated potato cells stained with 4′,6-diamidino-2-phenylindole (DAPI) showed chromatin condensation, and isolated DNA from DMS-treated cells had ladder pattern, confirming that DMS-induced plant cell death is a hypersensitive reaction-like programmed cell death. Involvement of ceramide signaling in induction of plant defense reactions is discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Ulocladium atrum</Emphasis> as a biological control agent for white mold of bean caused by<Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

Field studies were conducted near Lethbridge, Alberta, Canada, in 2001, 2004 and 2005 to determine the efficacy of the antagonistic fungusUlocladium atrum for control of white mold of bean caused bySclerotinia sclerotiorum. Results of the 3 years of field trials showed that, compared with the untreated control, foliar application of a spore suspension ofU. atrum (300 ml m⁻² of 10⁶ spores ml⁻¹ suspension) significantly reduced incidence and severity of white mold, increased seed yield and reduced contamination of bean seed by sclerotia ofS. sclerotiorum. The level of control of white mold observed in the treatment ofU. atrum was similar to that of the mycoparasitic fungusConiothyrium minitans, but lower than the fungicide treatments of Ronilan (vinclozolin) at the rate of 1200 g ha⁻¹ per application in 2001, or Lance (boscalid) at the rate of 750 g ha⁻¹ per application in 2004 and 2005. The potential for use ofU. atrum as a biological control agent for sclerotinia diseases is discussed. http://www.phytoparasitica.org posting Nov. 12, 2006.     

Note: Evaluation of antibacterial activity of essential oil of<Emphasis Type="Italic">Rosa damascena</Emphasis> on<Emphasis Type="Italic">Erwinia amylovora</Emphasis>

Significant antibacterial activity was observed in the essential oil (E.O.) ofRosa damascena Mill. and the Minimum Bactericidal Concentration (MBC) of the E.O. was determined as 1386.5 μg ml⁻¹ forErwinia amylovora, the causal agent of fire blight disease. Ooze formation on immature pears and lesion formation in artificially inoculated shoots were completely (100%) prevented by the essential oil ofR. damascena (1500 μg ml⁻¹), essential oil ofThymbra spicata var.spicata (500 μg ml⁻¹) and streptomycin (100 μg ml⁻¹). Copper oxychloride plus maneb significantly reduced ooze formation and lesion formation, but the control was less than that obtained with the essential oils or streptomycin. The essential oil ofR. damascena may be a useful natural bactericide for the control of the fire blight pathogen,E. amylovora. http://www.phytoparasitica.org posting July 14, 2004.     

<Emphasis Type="Italic">Eremothecium coryli</Emphasis> and <Emphasis Type="Italic">E.</Emphasis><Emphasis Type="Italic">ashbyi</Emphasis> cause yeast spot of azuki bean

Yeast-like fungi were isolated from lesions on azuki bean (cv. Shin-Kyotodainagon) seeds that had been sucked by bean bugs in Kyoto Prefecture, Japan. On the basis of morphological and physiological characteristics and sequence data of the internal transcribed spacer (ITS) regions including the 5.8S rDNA, these yeasts were identified as Eremothecium coryli and E. ashbyi. Pathogenicity of those yeasts was confirmed by a reinoculation test. To our knowledge, this is the first report of the occurrence of yeast spot in azuki bean in Japan. The nucleotide sequence data reported are available in the GeneBank/EMBL/DDBJ database as accessions AB478291–AB478309 for E. coryli AZC1–19 and AB478310–AB478317 for E. ashbyi AZA1–8.     

Pathogenicity,colony morphology and diversity of isolates of <Emphasis Type="Italic">Guignardia citricarpa</Emphasis> and <Emphasis Type="Italic">G. mangiferae</Emphasis> isolated from <Emphasis Type="Italic">Citrus</Emphasis> spp.

In the present study, the pathogenicity of 36 isolates of Guignardia species isolated from asymptomatic ‘Tahiti’ acid lime fruit peels and leaves, ‘Pêra-Rio’ sweet orange leaves and fruit peel lesions, and a banana leaf were characterized. For pathogenicity testing, discs of citrus leaves colonized by Phyllosticta citricarpa under controlled laboratory conditions were kept in contact with the peels of fruit that were in susceptible states. In addition, pathogenicity was related to morphological characteristics of colonies on oatmeal (OA) and potato dextrose agar (PDA). This allowed the morphological differentiation between G. citricarpa and G. mangiferae. Polymerase chain reactions (PCRs) were also used to identify non-pathogenic isolates based on primers specific to G. citricarpa. A total of 14 pathogenic isolates were detected during pathogenicity tests. Five of these were obtained from leaf and fruit tissues of the ‘Tahiti’, which until this time had been considered resistant to the pathogen. Given that the G. citricarpa obtained from this host was pathogenic, it would be more appropriate to use the term insensitive rather than resistant to categorize G. citricarpa. A non-pathogenic isolate was obtained from lesions characteristic of citrus black spot (CBS), indicating that isolation of Guignardia spp. under these conditions does not necessarily imply isolation of pathogenic strains. This also applied to Guignardia spp. isolates from asymptomatic citrus tissues. Using fluorescent amplified fragment length polymorphism (fAFLP) markers, typically pathogenic isolates were shown to be more closely related to one another than to the non-pathogenic forms, indicating that the non-pathogenic isolates display higher levels of genetic diversity.     

Pathogenicity of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> against <Emphasis Type="Italic">Galleria mellonella</Emphasis>

The greater wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) is occasionally found in beehives and is a major pest of stored wax. Entomopathogenic fungi have recently received attention as possible biocontrol elements for certain insect pests. In this study, 90 isolates of Beauveria bassiana and 15 isolates of Metarhizium anisopliae were screened for proteases and lipases production. The results showed significant variations in the enzymatic action between the isolates. In the bioassay, the selected isolates evinced high virulence against the 4th instar of the G. mellonella larvae. The isolates BbaAUMC3076, BbaAUMC3263 and ManAUMC3085 realized 100% mortality at concentrations of 5.5 × 10⁶ conidia ml⁻¹, 5.86 × 10⁵ conidia ml⁻¹, and 4.8 × 10⁶ conidia ml⁻¹, respectively. Strong enzymatic activities in vitro did not necessarily indicate high virulence against the tested insect pest. The cuticle of the infected larvae became dark and black-spotted, indicating direct attack of fungus on the defense system of the insects. The LC₅₀ values were 1.43 × 10³, 1.04 × 10⁵ and 5.06 × 10⁴ for Bba3263AUMC, Bba3076AUMC and Man3085AUMC, respectively, and their slopes were determined by computerized probit analysis program as 0.738 ± 0.008, 0.635 ± 0.007 and 1.120 ± 0.024, respectively.     

Genetic variation among <Emphasis Type="Italic">Fusarium</Emphasis> isolates from onion,and resistance to <Emphasis Type="Italic">Fusarium</Emphasis> basal rot in related <Emphasis Type="Italic">Allium</Emphasis> species

The aim of this research was to study levels of resistance to Fusarium basal rot in onion cultivars and related Allium species, by using genetically different Fusarium isolates. In order to select genetically different isolates for disease testing, a collection of 61 Fusarium isolates, 43 of them from onion (Allium cepa), was analysed using amplified fragment length polymorphism (AFLP) markers. Onion isolates were collected in The Netherlands (15 isolates) and Uruguay (9 isolates), and received from other countries and fungal collections (19 isolates). From these isolates, 29 were identified as F. oxysporum, 10 as F. proliferatum, whereas the remaining four isolates belonged to F. avenaceum and F. culmorum. The taxonomic status of the species was confirmed by morphological examination, by DNA sequencing of the elongation factor 1-α gene, and by the use of species-specific primers for Fusarium oxysporum, F. proliferatum, and F. culmorum. Within F. oxysporum, isolates clustered in two clades suggesting different origins of F. oxysporum forms pathogenic to onion. These clades were present in each sampled region. Onion and six related Allium species were screened for resistance to Fusarium basal rot using one F. oxysporum isolate from each clade, and one F. proliferatum isolate. High levels of resistance to each isolate were found in Allium fistulosum and A. schoenoprasum accessions, whereas A. pskemense, A. roylei and A. galanthum showed intermediate levels of resistance. Among five A. cepa cultivars, ‘Rossa Savonese’ was also intermediately resistant. Regarding the current feasibility for introgression, A. fistulosum, A. roylei and A. galanthum were identified as potential sources for the transfer of resistance to Fusarium into onion.